Antibiotic formulation and use for drug resistant infections

ABSTRACT

A liposomal aminoglycoside formulation comprising a neutral lipid, a negatively charged lipids and a sterol. The formulation contains unilamellar vesicles having an average size below 100 nm. A process of making liposomes containing an aminoglycoside is provided where the hydration temperature is significantly below the transition temperature of the formulation. A method for the treatment of drug susceptible and drug resistant bacteria.

This is a 371 of PCT/US93/04501, filed May 11, 1993, which is aContinuation-in-Part application of PCT/US92/10591, filed Dec. 2, 1992.

FIELD OF THE INVENTION

This invention relates to the fields of biochemistry and medicine, andparticularly to a liposome formulation. More specifically, it relates toa liposomal formulation containing an aminoglycoside, its process ofmanufacture and its use. This invention also relates to formulationshaving reduced toxicity, longer stability, and superior efficacy. Thisinvention further relates to liposomal formulations containing amikacinand its use in treating drug susceptible and drug resistant strains ofbacterial infections.

BACKGROUND OF THE INVENTION

The discovery of aminoglycosides began in the 1940s with the isolationof streptomycin from Streptomyces griseus. Since the 1940s, otheraminoglycosides have been discovered and synthesized. These includeneomycin which is obtained or isolated from Streptomyces fradiae;kanamycin which is isolated from Streptomyces kanamyceticus; gentamicinwhich is isolated from Micromonospora purpurea; tobramycin which isisolated from Streptomyces tenedrarius; sisomicin isolated frommicromonospora inyoesis; amikacin which is a semisynthetic derivative ofKanamycin A; and netilmicin which is a semisynthetic derivative ofsisomicin. Amikacin has the broadest spectrum of antimicrobial activityof all the aminoglycosides. It also has a unique resistance to theimmunoglycoside-inactivating enzymes.

The aminoglycosides are polar-cations which consist of two or more aminosugars joined in a glycosidic linkage to a hexose nucleus, which isusually in a central position. The aminoglycosides are used primarily totreat infections caused by gram-negative bacteria. However,aminoglycosides have been used in recent years to treat bacteria fromthe genera Mycobacteria. For example, amikacin has shown to be effectiveagainst Mycobacterium tuberculosis. Aminoglycosides have also beentested against M.avium infections including M. avium-intracellularecomplex (MAC) which is a group of related acid-fast organisms that growonly slightly faster than M. tuberculosis and can be divided into anumber of serotypes. At the beginning of the twentieth century,tuberculosis was the most prevalent cause of death in the United States.By the late 1940s, with the advent of streptomycin, tuberculosisinfection had decreased substantially. Since the mid-1980s with theappearance of the acquired immune deficiency syndrome, tuberculosisagain began to emerge as a major health problem. Further, the new casesof tuberculosis showed resistance to many of the available antibiotictherapies. Similarly MAC, once considered rare, is now the most commonsystemic bacterial type infections in patients suffering from acquiredimmune deficiency syndrome. Hence, the search for an effectiveantibiotic has intensified.

Although the aminoglycosides have been useful in treating infections,the use of these antibiotics is not free from toxicity and side effects.Aminoglycosides may produce irreversible vestibular, cochlear, and renaltoxicity. The two main toxic effects of aminoglycosides are ototoxicityand nephrotoxicity. Studies have found that the aminoglycosidesantibiotics may cause polyuria, decreased urinary osmolality,proteinuria, enzymuria, glycosuria, and a decrease in the rate ofglomerular filtration. Some investigators believe that nephrotoxicityresults from the accumulation of the aminoglycosides in the renal cortexbecause of the long half-life of the agents in that tissue.

Liposomes are microscopic vesicles made, in part, from phospholipidswhich form closed, fluid filled spheres when dispersed with water.Phospholipid molecules are polar, having a hydrophilic ionizable headand two hydrophobic tails consisting of long fatty acid chains. Thus,when sufficient phospholipid molecules are present with water, the tailsspontaneously associate to exclude water while the hydrophilic phosphateheads interact with water. The result is a bilayer membrane in which thefatty acid tails converge in the newly formed membrane's interior andthe polar heads point in opposite directions toward an aqueous medium.These bilayer membranes can be caused to form closed spheres known asliposomes. The polar heads at the inner surface of the membrane pointtoward the aqueous interior of the liposome. At the opposite surface ofthe spherical membrane, the polar heads interact with the surroundingaqueous medium. As the liposomes are formed, water soluble molecules canbe incorporated into the aqueous interior, and lipophilic molecules willtend to be incorporated into the lipid bilayer. Liposomes may be eithermultilamellar, like an onion with liquid separating many lipid bilayers,or unilamellar, with a single bilayer surrounding an entirely liquidcenter.

There are many types of liposome preparation techniques which may beemployed and which produce various types of liposomes. These can beselected depending on the use, the chemical intended to be entrapped,and the type of lipids used to form the bilayer membrane. Therequirements which must be considered in producing a liposomepreparation are similar to those of other controlled release mechanisms.They are as follows: (1) high percent of chemical entrapment; (2)increased chemical stability; (3) low chemical toxicity; (4) rapidmethod of production; and (5) reproducible size distribution.

The first method described to encapsulate chemicals in liposomesinvolved production of multilamellar vesicles (MLVs). Methods forencapsulating chemicals in MLVs are known in the art.

Liposomes can also be formed as unilamellar vesicles (UVs), whichgenerally have a size less than 1 μm. There are several techniques knownin the art which are used to produce unilamellar liposomes.

Smaller unilamellar vesicles can be formed using a variety oftechniques. By dissolving phospholipids in ethanol and injecting theminto a buffer, the lipids will spontaneously rearrange into unilamellarvesicles. This provides a simple method to produce UVs which haveinternal volumes similar to that of those produced by sonication(0.2-0.5 L/mol/lipid). Sonication or extrusion (through filters) of MLVsalso results in dispersions of UVs having diameters of up to 0.2 μm,which appear as clear or translucent suspensions.

Another common method for producing small UVs is the detergent removaltechnique. Phospholipids are solubilized in either ionic or non-ionicdetergents such as cholates, Triton X, or n-alkylglucosides. The drug isthen mixed with the solubilized lipid-detergent micelles. Detergent isthen removed by one of several techniques: dialysis, gel filtration,affinity chromatography, centrifugation, ultrafiltration. The sizedistribution and entrapment efficiencies of the UVs produced this waywill vary depending on the details of the technique used.

The therapeutic use of liposomes includes the delivery of drugs whichare normally toxic in the free form. In the liposomal form the toxicdrug may be directed away from the sensitive tissue and targeted toselected areas. Liposomes can also be used therapeutically to releasedrugs, over a prolonged period of time, reducing the frequency ofadministration. In addition, liposomes can provide a method for formingan aqueous dispersion of hydrophobic drugs for intravenous delivery.

When liposomes are used to target encapsulated drugs to selected hosttissues, and away from sensitive tissues, several techniques can beemployed. These procedures involve manipulating the size of theliposomes, their net surface charge as well as the route ofadministration. More specific manipulations have included labeling theliposomes with receptors or antibodies for particular sites in the body.

The route of delivery of liposomes can also affect their distribution inthe body. Passive delivery of liposomes involves the use of variousroutes of administration, e.g., intravenous, subcutaneous and topical.Each route produces differences in localization of the liposomes. Twocommon methods used to actively direct the liposomes to selected targetareas are binding either antibodies or specific receptor ligands to thesurface of the liposomes. Antibodies are known to have a highspecificity for their corresponding antigen and have been shown to becapable of being bound to the surface of liposomes, thus increasing thetarget specificity of the liposome encapsulated drug.

Since the chemical composition of many drugs precludes their intravenousadministration, liposomes can be very useful in adapting these drugs forintravenous delivery. Many hydrophobic drugs, including cyclosporine,fall into this category because they cannot be easily dissolved in awater-based medium and must be dissolved in alcohols or surfactantswhich have been shown to cause toxic reactions in vivo. Liposomes,composed of lipids, with or without cholesterol, are nontoxic.Furthermore, since liposomes are made up of amphipathic molecules, theycan entrap hydrophilic drugs in their interior space and hydrophobicmolecules in their lipid bilayer. Although methods for making liposomesare well known in the art, it is not always possible to determine aworking formulation without undue experimentation.

Liposomal formulations containing aminoglycosides have been prepared.Many of the preparations include aerosol formulations using MLVs. Otherformulations contain a large amount of negatively charged lipids,generally is greater than 20%, to increase retention time or circulationhalf-life. Problems associated with aminoglycosides liposomalformulations include short retention time in the system because of RESuptake. Attempted formulations in the art have also resulted inliposomal aminoglycosides that are unstable--both on the shelf and inserum.

Thus, it is a desideratum to provide for a novel formulation which wouldincrease the retention time of an aminoglycoside in a mammal's systemand which can deliver more drug with superior efficacy and lowertoxicity then free drug. It is also desirable to provide a process whichallows the manufacture of a clear, stable and efficacious aminoglycosideliposome suspension because of the inability of those of ordinary skillto produce a therapeutically effective aminoglycoside unilamellarliposomal formulation having an average particle size of less than 100nm, preferably with a high drug to total lipid ratio.

SUMMARY OF THE INVENTION

Liposomes are provided in the present invention which comprise anaminoglycoside wherein the liposomes are unilamellar having an averagesize of 100 nm or less. A liposomal formulation is provided whichcomprises an aminoglycoside wherein the liposomes are comprised of aneutral lipid, a sterol and a negatively charged lipid. Smallunilamellar vesicles are also provided wherein the molar amount ofnegatively charged lipid is less than 20% of total lipid. A preferredformulation is liposomes having an aminoglycoside wherein the liposomesare unilamellar vesicles having an average size of 30 nm to 100 nm andfurther comprised of a phosphatidylcholine, cholesterol, and aphosphatidylglycerol wherein the molar amount of phosphatidylglycerol isless than 5% and preferably about 3%. The drug to total lipid ratio isbetween 1:9 and 1:3 with the preferred ratio at about 1:4. A preferredformulation is also provided comprising liposomes including anaminoglycoside wherein the liposomes are unilamellar vesicles having anaverage size less than 100 nm wherein the lipids comprise hydrogenatedsoy phosphatidylcholine, cholesterol, and distearoylphosphatidylglycerolin a molar ratio of about 2:1:0.1.

The present invention also includes a method for making aminoglycosideliposomes. The process comprises forming a lipid powder comprised of aneutral phospholipid, a sterol, and a negatively charged lipid; mixingthe powder with an aminoglycoside in an aqueous buffer and hydrating themixture at a temperature significantly below the transition temperatureof the lipid mixture; and reducing the size of the liposome to anaverage particle size of less than 100 nm.

The present invention also provides for the treatment of bacterialinfections in mammals comprising preparing a liposomal formulationhaving an aminoglycoside wherein the liposomes are those described aboveand which are used to treat infections by introducing a therapeuticeffective amount of the liposomes into a mammal. Thus, the presentinvention provides the use of liposomal aminoglycoside formulations totreat bacterial infections. The bacterial infections treated includeopportunistic aerobic gram-negative bacilli such as the generaPseudomonas. Another aspect of the invention includes the use of theliposomal formulations in the treatment of a bacterial infection causedby P. aeruginosa. The method of treating bacterial infections is notlimited to gram-negative infections. The liposomal formulations can beused to treat bacterial infections comprising gram-positive bacilli suchas the genera Mycobacterium. The invention is particularly useful fortreating mycobacterium which causes tuberculosis-like diseases. Numerousbacterium may be treated using the liposomes described above. Thebacteria would include: M. tuberculosis, M. leprae, M. Intracellulare,M. smegmatis, M. bovis, M. kansasii, M. avium, M. scrofulcium, or M.africanum. Liposomal formulations of aminoglycoside are alsoparticularly useful in treating MAC.

A particularly useful aspect of the invention is a method of treating adrug resistant bacterial infection in a patient, comprising the deliveryto the patient an effective amount of liposomes comprising anencapsulated aminoglycoside wherein the liposomes are comprised ofunilamellar vesicles comprised of a neutral lipid, cholesterol and anegatively charged lipid, and having an average size of less than 100nm. Experiments performed in vitro establish that liposomal amikacininhibits and kills drug resistant M. tuberculosis. The experimentsperformed further establish that liposomal amikacin kills M.tuberculosis whereas the free drug, at equivalent dosage concentration,only inhibits the growth of the bacteria. Killing is defined as areduction in the number of colony forming units of bacteria from aprevious time point. Inhibition is defined as an increase in, or thesame number of, colony forming units of bacteria from a previous timepoint but less than the number of colony forming units shown foruntreated cultures at the same time points. Thus, the present inventionprovides for the killing of the bacteria at tolerable non-toxic levelsin cases where the bacteria is resistant to aminoglycosides and otherantibiotics or where the free drug has at the most an inhibitory effect.

The present invention also shows that liposomal amikacin is retained inblood plasma significantly longer than free amikacin. Intermittenttreatment of non-compliant patients is obtained in the present inventionas the present invention provides higher peak serum levels, prolongedserum half life and increased uptake and retention by macrophages.

The present invention also provides a method for the treatment of drugsusceptible M. tuberculosis by delivering an effective amount ofliposomal amikacin to a patient wherein the dosage levels provideinhibition or killing at levels equivalent to or greater than freeamikacin. Thus advantages provided by the decreased toxicity andincreased serum levels of amikacin delivered by liposomal amikacinprovide a preferable and useful alternative to treatment provided by thefree (unencapsulated) amikacin.

Many, if not all, mycobacterium infections discussed above are difficultto treat because the bacteria invade phagocytic cells such asmacrophages. Application of liposomal formulations containing anaminoglycoside result in the intracellular delivery of the drug whichwould not normally occur with the delivery of the free drug. Thus, thepresent invention provides a treatment of infected phagocytic cells inmammals by delivering a therapeutic or effective amount of anaminoglycoside into a macrophage using a unilamellar liposome having anaverage size of 100 nm or less.

Provided herein is a liposomal amikacin formulation which can bedelivered to a mammal and which provides the following benefits overfree amikacin: 1) significantly higher doses of amikacin delivered tosites of infection; 2) substantially lower toxicity; and 3) retention inblood plasma for a significantly longer period of time.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term liposome refers to unilamellar vesicles ormultilamellar vesicles such as described in U.S. Pat. Nos. 4,753,788 and4,935,171, the contents of which are incorporated herein by reference.

The present invention provides liposomal aminoglycoside formulationpreferably containing a neutral lipid such as a phosphatidylcholine, aphosphatidylglycerol, cholesterol (CHOL) and amikacin. Preferred lipidinclude lipids which are chemically pure and/or are fully saturated. Thepreferred neutral lipids are saturated lipids such as hydrogenated eggphosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC),distearoyl phosphatidylcholine (DSPC), and dipalmitoylphosphatidylcholine (DPPC). The preferred carbon chain lengths of theneutral lipids are from C₁₆ -C₁₈. The preferred negatively chargedlipids are saturated lipids such as hydrogenated soy phosphatdylglycerol(HSPG), hydrogenated egg phosphatidylglycerol (HEPG),distearyolphosphatidylglycerol (DSPG), dimyristoylphosphatidylcholine.Hydrogenated soy phosphatidylcholine (HSPC),distearoylphosphatidylglycerol (DSPG) are the preferred lipids for usein the invention. Other suitable phosphatidylcholines include thoseobtained from egg or plant sources, or those that are partially orwholly synthetic. Other phosphatidylglycerols that may be used aresaturated semisynthic lipids having carbon chain lengths from C₁₂ -C₁₈and include dimyristoyl phosphatidylglycerol (DMPG) anddilaurylphosphatidylglycerol (DLPG).

The preferred formulation includes liposomes comprising an encapsulatedaminoglycoside wherein the liposomes are unilamellar vesicles having anaverage size of less than 100 nm and which are stable for at least twoweeks at 22° C. without a significant change in size or without loss ofmore than 10% of encapsulated aminoglycosides. Generally the size of theliposomes would not vary by more than 30% and most preferably would notvary by more than 20%. The preferred ratio of HSPC:CHOL:DSPG is about2:1:0.1 and the drug to total lipid ratio is about 1:4. Other preferredformulations include DSPG in a molar amount of 0 to 20% and mostpreferably in a molar amount of less than 5%. Other preferredformulations include formulations where the drug to total lipid ratio isfrom 1:9 to 1:3.

The process of the present invention is initiated with the preparationof a solution from which the liposomes are formed. A quantity of aphosphatidylcholine, a phosphatidylglycerol and cholesterol is dissolvedin an organic solvent, preferably a mixture a 1:1 (by volume) mixture ofchloroform and methanol, to form a clear solution. Other solvents (andmixture thereof), such as ether, ethanol and other alcohols can be used.The preferred temperature to dissolve the lipids is between roomtemperature and 60° C., preferably at room temperature. The solution isevaporated to form a lipid film or a lipid powder. To form a lipid film,the solvents are evaporated under nitrogen between room temperature and60° C., preferably at room temperature. To form a lipid powder, themixture of lipids in solution as described above is sprayed in a spraydrier. Preferably, the spraying takes place under nitrogen.

An aminoglycoside, for example, amikacin free base, is dissolved in anaqueous phosphate buffer with 9% sucrose and the pH is adjusted between6 and to 8, preferably between 6 and 7.5 and most preferably between 6.2and 6.6. The preferred pH is about 6.4. The buffer may also be adjustedto a pH of about 7.4. The pH of the buffer is adjusted with dilutedacids and bases preferably with 6N HCI and 2.5N NaOH. The preferredbuffer is 10 mM phosphate buffer. However, other aqueous buffers can beused such as succinate buffer (Disodium succinate hexahydrate). Theaminoglycoside solution is mixed with either the lipid film or the lipidpowder and hydrated, preferably between 40° C. and 65° C. mostpreferably between 45° C. and 65° C. The solution should be hydrated forat least ten minutes.

Unilamellar vesicles are formed by the application of a shearing forceto the hydrated solution, e.g., by extrusion, sonication, or the use ofa homogenizing apparatus such as a Gaulin homogenizer or a French press.Shearing force can also be applied using injection, freezing andthawing, dialyzing away a detergent solution from lipids, or other knownmethods used to prepare liposomes. The size of the liposomes can becontrolled using a variety of known techniques including the duration ofshearing force. Preferably, the modified Gaulin homogenizing apparatusdescribed in U.S. Pat. No. 4,753,788 is employed to form unilamellarvesicles having diameters of less than 100 nm at a pressure of 7,000 to13,000 psi and a temperature significantly below the transitiontemperature of the lipids.

The above described formulations are particularly useful for thetreatment of MAC and Pseudomonas aeruginosa infections. The use of theabove formulations indicate that MAC may be treated with significantlymore amikacin delivered in a liposomal formulation than with free drug.For example it has been shown that up to 320 mg of amikacin per kilogramof mice body-weight which is more than 50% more than is tolerated withfree drug. The use of the above formulations also indicates that aliposomal formulation is able to deliver significantly more amikacin toa mammal than free drug in the treatment of P. aeruginosa.

Amikacin delivered through a liposomal formulation is also retained inthe plasma longer than free amikacin.

The above described formulations are also efficacious in inhibiting andkilling both drug resistant and drug susceptible M. tuberculosis asestablished by in vitro testing. In one experiment the drug resistantstrain Vertulla of M. tuberculosis was tested. In another experiment thedrug susceptible strain H37RV was tested. The experiments were carriedout as described in Example 6. Briefly, human monocytes derivedmacrophage cultures were developed and infected with either the drugresistant strain or the drug susceptible strain. Liposomal amikacin wasprepared as described in Example 1 below. The liposomes comprised HSPC,cholesterol and DMPG in a molar ration of about 2:1:0.1. The drug tototal lipid ratio was 1:4 (about 25%). The average size of the liposomeswas under 100 nm.

In the experiment performed on the susceptible strain, liposomal andfree amikacin were added to the cultures at the following concentrations(μg/ml): 1, 2, and 4. Untreated cultures and cultures treated withliposomes-only were used as controls. The cultures were assayed at 0, 4,and 7 days after infection. The 1 μg/ml concentrations of both the freeamikacin and the liposomal amikacin inhibited the growth of the bacteriawith the liposomal amikacin showing greater inhibition and also killingat day 4. The 2 μg/ml concentrations of both the free amikacin and theliposomal amikacin were also effective in treating the infection whereinthe liposomal amikacin obtained killing at day 4 and the free drugobtained inhibition. The 4 μg/ml concentrations of both the liposomaland the free amikacin obtained killing at day 4 with the liposomalamikacin showing greater killing. Thus, liposomal amikacin provides apreferred treatment of M. tuberculosis because the present inventionprovides, at the least, equivalent inhibition and killing at similarconcentrations than free amikacin with lower toxicity and longer plasmaretention time.

In the experiment performed on the resistant strain, liposomal and freeamikacin were added to the cultures at the following concentrations(μg/ml): 4, 8, and 16. Untreated cultures and cultures treated withliposomes-only were used as controls. The cultures were assayed at 0, 4,and 7 days after infection. At day 4 the liposomal amikacin achieved abacterial growth of a least twenty-one-fold, sixty-four-fold and severalhundred-fold (e.g. 563×) less than the untreated control for the 4μg/ml, 8 μg/ml and 16 μg/ml concentration, respectively. This activitywas 8.7, 12.9 and 130 times more effective than for the respective freeamikacin concentrations.

At day 7 the liposomal amikacin achieved bacterial growth of at leastone-hundred-fold, (e.g. 133×), one-thousand-fold (e.g. 1110×) andseveral thousand-fold (e.g. 5880×) less than the untreated control. Thisactivity was 41, 11.9 and 46 times more effective than for therespective amikacin concentrations.

The results show that free amikacin would only inhibit the growth of theinfection without killing at the concentrations tested. The liposomalamikacin provided killing of the drug resistant bacteria at allconcentrations at day 4. Thus, liposomal amikacin provided high degreeof killing where only an inhibitory effect was expected against the drugresistant strain.

Since dosage regimens for aminoglycosides are well known to medicalpractitioners, the amount of the liposomal aminoglycoside formulationwhich is effective for the treatment of infections in mammals andparticularly humans will be apparent to those skilled in the art.

This invention will be more fully understood by reference to thefollowing examples, which are intended to be illustrative of theinvention, but not limiting thereof. Examples 1-6 detail the formation,and both chemical and biological testing of the liposomal amikacinformulations of the invention.

EXAMPLE 1

A lipid mixture of hydrogenated soy PC, cholesterol, and distearoylphosphatidylglycerol was provided in a molar ratio of 2:1:0.1respectively. The lipids were dissolved in chloroform and methanol (1:1by volume). The resulting solution was stirred until the lipidsdissolved and a clear solution was formed. The mixing is best carriedout at room temperature. A lipid film was obtained by evaporating theorganic solvents under Nitrogen at room temperature.

Amikacin free base was dissolved in 10 mM phosphate buffer with 9%sucrose and the pH adjusted to 7.4 with 6.0M HCl. The lipid film and theamikacin solution were mixed so that the concentration of the drug wasabout 250 mg/ml and the concentration of the lipid was about 100 mg/ml.The resulting solution was stirred and hydrated for 10-15 minutes at 65°C. The solution was sonicated for 20 minutes using a probe sonicator(Model 500 Sonic & Material). The solution was held at 65° C. for tenminutes. The solution was cooled to room temperature and centrifuged at3600 rpm for 10 minutes. The supernatant was collected and pouredthrough a Sephadex G-50 column to separate the liposomal formulationfrom the free drug. The concentration of amikacin and lipid componentswere determined by HPLC assay. The size was determined by opticalparticle sizing. The size of the liposomes varied from experiment toexperiment from about 40-100 nm. For example, in one experiment a meandiameter of 62.1 nm was observed. In another, a mean diameter of 47.4 nmwas observed. A preferred formulation contains a drug to lipid ratio of1:4.

EXAMPLE 2

A lipid solution was prepared in methanol and chloroform as described inExample 1. A lipid powder was obtained in a spray drier (Yamato PulvisBasic Unit, Model GB-21). The following settings were used: 1) pump dialat 3-4.5; 2) aspirator dial at 6; 3) pressure at 1.5-2 Kgf/cm² s; 4)inlet temperature at 50° C.; and 5) outlet temperature at 40° C. Thespraying took place under nitrogen for two hours. The powder wascollected and combined with amikacin drug solution (as prepared inExample 1). The resulting solution was mixed for 2 minutes using a highshear mixer (Virtishear)) at 1000 rpm. The solution was placed in abeaker and set in a 40° C. water bath and hydrated with mixing until thesolution reached 40° C. (about 25 minutes). The solution was then placedin a homogenizer (Gaulin 15M) for approximately 30 passes at 10,000 psiwhile maintaining the inlet temperature at 40° C. The resulting solutionwas filtered through a 0.8 micron nylon filter. The solution wasultrafiltered to replace unencapsulated drug with new buffer. Thesolution was washed with 7 to 10 volumes of buffer. The resultingproduct was heated to 40° C. and filtered through successive 0.8, 0.45,and 0.22 micron (pore size) filters. Thus, a surprising aspect of thepresent invention is that the hydration of liposomes occurredsignificantly below the transition temperature of the formulation (about52° C.).

EXAMPLE 3

The testing of liposome encapsulated amikacin for the treatment of MACwas performed using a murine model. Beige mice (C57B1/6bg^(j) /bg^(j))were infected with MAC (101, type 1). The mice were infected byinjection of 1×10⁷ Colony Forming Units (cfu) in the mouse tail vein(i.v.). Three experiments were performed. In the first experiment, 40,80 and 120 milligrams of amikacin (liposomal and free) per kilogram ofmouse body-weight was given i.v. daily for 5 days, beginning 7 daysafter infection. The animals were sacrificed 5 days after treatment wascompleted and the liver, lung and spleen tissue were plated.Quantitation of organisms in liver, spleen and lung tissue wasperformed. The cfu were determined by growth on Middlebrook 7h11 agarplates. Untreated and empty liposomes were used as controls.

The liposomes were prepared as in Example 1. This experiment wasperformed in two identical parts. The liposomes in the first part wereof an average size of 49.8 nm and the average size of the liposomes inthe second part were 73.7 nm (mean diameter). The amikacin concentrationwas either 15.0 mg/ml or 13.21 mg/ml and the total lipid concentrationwas either 121 mg/ml or 55.7 mg/ml respectively (drug to lipid ratios:0.123, 0.24). Free drug for all experiments was prepared in the samebuffer that the liposomal formulations were contained in. The resultsare listed in Table 1. Table 1 lists the results for the spleen andliver tissue.

                  TABLE 1                                                         ______________________________________                                        Effects of Liposomal Amikacin on MAC Infected Mice Tissue                             #    Spleen        Liver                                              Regimen   mice   cfu/g    log cfu/g                                                                            cfu/g  log cfu/g                             ______________________________________                                        untreated 6      1.13 × 10.sup.9                                                                  9.05   7.49 × 10.sup.8                                                                8.87                                  liposomes only                                                                          3      5.30 × 10.sup.8                                                                  8.72   5.51 × 10.sup.8                                                                8.74                                  amikacin 40 mg/                                                                         5      4.08 × 10.sup.8                                                                  8.61   1.41 × 10.sup.8                                                                8.15                                  kg                                                                            amikacin 80 mg/                                                                         6      4.82 × 10.sup.8                                                                  8.68   7.79 × 10.sup.7                                                                7.89                                  kg                                                                            amikacin 120 mg/                                                                        3      3.46 × 10.sup.8                                                                  8.54   5.65 × 10.sup.7                                                                7.75                                  kg                                                                            liposomal 6      5.43 × 10.sup.7                                                                  7.73   1.44 × 10.sup.7                                                                7.16                                  amikacin 40 mg/                                                               kg                                                                            liposomal 6      2.71 × 10.sup.7                                                                  7.43   2.11 × 10.sup.7                                                                7.32                                  amikacin 80 mg/                                                               kg                                                                            liposomal 6      4.03 × 10.sup.7                                                                  7.61   9.86 × 10.sup.6                                                                6.99                                  amikacin 120 mg/                                                              kg                                                                            ______________________________________                                    

The results for the lung study were as follows: 1) untreated regimenwere 2.86×10⁷ cfu/gram (log=7.46);2) empty liposomes were 2.95×10⁷cfu/gram (log=7.47);3) free amikacin (40 mg/kg, 80 mg/kg and 120 mg/kg)were 1.44×10⁶ (log=6.16), 1.29×10⁶ (log 6.11) and 9.94×10⁵ (log=6.00)respectively; 4) liposomal amikacin (40 mg/kg, 80 mg/kg, and 120 mg/kg)were 2.28×10⁵ (log=5.36), 3.02×10⁵ (log=5.48) and 4.15×10⁵ (log=5.62).

Another experiment was carried out as above (also in two parts) exceptthat the drug therapy was started 5 days after infection. The drug wasgiven i.v. three times/week for 21 days. The mice were sacrificed 1-2days after the treatment stopped. During the treatment period, a groupof mice received 40 mg/Kg, 80 mg/kg, and 150 mg/kg of free amikacin andanother group received 40 mg/kg, 80 mg/kg, and 160 mg/kg of liposomalamikacin. The lung tissues were not tested. Similar controls were usedas above. The liposomes were prepared as described in Example 1. Theaverage size of the liposomes was 66.5 nm or 79.6 nm (mean diameter).The concentration of the amikacin was either 15.98 mg/ml or 6.74 mg/ml.The total lipid was measure only the second part of the experiment andit was 27.0 mg/ml (drug to lipid ratio: 1:4.167). The results are listedin Table 2.

                  TABLE 2                                                         ______________________________________                                        Effects of Liposomal Amikacin on MAC Infected Mice Tissue                                  Spleen     Liver                                                 Regimen    # mice  cfu/g    log cfu                                                                             cfu/g  log cfu                              ______________________________________                                        untreated  6       2.23 × 10.sup.9                                                                  9.35  1.06 × 10.sup.9                                                                9.03                                 liposomes only                                                                           5       2.27 × 10.sup.9                                                                  9.36  2.08 × 10.sup.9                                                                9.32                                 amikacin 40 mg/kg                                                                        6       5.76 × 10.sup.8                                                                  8.76  1.09 × 10.sup.8                                                                8.04                                 amikacin 80 mg/kg                                                                        6       2.02 × 10.sup.8                                                                  8.31  3.67 × 10.sup.7                                                                7.56                                 amikacin 120 mg/kg                                                                       2       1.48 × 10.sup.8                                                                  8.17  3.15 × 10.sup.7                                                                7.50                                 liposomal amikacin                                                                       6       7.98 × 10.sup.7                                                                  7.90  7.24 × 10.sup.6                                                                6.86                                 40 mg/kg                                                                      liposomal amikacin                                                                       4       2.57 × 10.sup.7                                                                  7.41  2.21 × 10.sup.6                                                                6.34                                 80 mg/kg                                                                      liposomal amikacin                                                                       6       1.73 × 10.sup.7                                                                  7.24  1.04 × 10.sup.6                                                                6.02                                 160 mg/kg                                                                     ______________________________________                                    

A third experiment was carried out as in the second experiment exceptthat the mice treated with the liposomal amikacin received 120 mg/kg,240 mg/kg and 320 mg/kg doses and the mice treated with the freeamikacin received only 120 mg/kg doses. The liposomes were prepared asin Example 2 where the average size of the liposomes were 81.8 nm(median diameter). The amikacin concentration was 32.02 mg/ml and thetotal lipid concentration was 91.5 mg/ml (drug to lipid ratio: 1:2.941).The results are listed in Table 3.

                                      TABLE 3                                     __________________________________________________________________________    Effects of Liposomal Amikacin on MAC Infected Mice Tissue                                  Spleen      Liver                                                Regimen  # mice                                                                            cfu/g  log cfu/g                                                                          cfu/g  log cfu/g                                     __________________________________________________________________________    untreated                                                                              6   3.70 × 10.sup.9                                                                9.57 1.65 × 10.sup.9                                                                9.22                                          liposomes only                                                                         6   4.05 × 10.sup.8                                                                8.61 1.20 × 10.sup.8                                                                8.08                                          amikacin 120 mg/kg                                                                     6   2.13 × 10.sup.8                                                                8.33 6.10 × 10.sup.7                                                                7.78                                          liposomal amikacin                                                                     6    8.8 × 10.sup.6                                                                6.94 3.67 × 10.sup.5                                                                5.56                                          120 mg/kg                                                                     liposomal amikacin                                                                     6   5.52 × 10.sup.6                                                                6.74 4.83 × 10.sup.5                                                                5.68                                          240 mg/kg                                                                     liposomal amikacin                                                                     6   <3.38 × 10.sup.6                                                               6.53 <2.00 × 10.sup.5                                                               5.30                                          320 mg/kg                                                                     __________________________________________________________________________

The results of all the experiments establish that significantly higherdoses of amikacin can be delivered without an increase in toxicity andwith superior efficacy. It is known in the art that 150 mg/kg ofamikacin will kill many of the mice injected. Thus, the delivery of 320mg/kg of amikacin is a significant increase in the amount of drugdelivered without lethal toxicity.

EXAMPLE 4

The efficacy and toxicity of liposomal amikacin in Pseudomonas infectedmice was tested. CF-1 mice were used (females, 6-8 weeks old). The micewere obtained from Jackson Labs. A clinical isolate of Pseudomonasaeruginosa was obtained on a Mueller Hinton/MacConkey blood agar plate.The organisms were transferred to a Mueller Hinton plate and grown for24 hours. Colonies were transferred to saline and grown for 48 hours.The samples were frozen in saline containing 15% fetal calf serum at1×10⁸ organisms/ml (MacFarland Standard). The colonies were stored at-70° C. The colonies were thawed and grown in a Mueller Hinton plate for24 hours. The bacteria were adjusted to 8×10⁶ organisms/ml in salinecontaining talc (62.5 mg/ml) and 1 ml was injected intraperitoneal. Aculture of inoculum on MH agar for 24 hours determined thatapproximately 7×10⁶ cfu were delivered per mouse. Liposomal amikacin andempty liposomes were prepared as set out in Example 2. The liposomes ofthe amikacin formulation had an average size (median diameter) of 62.4nanometers. The lipid concentration was 95.31 mg/ml and the amikacinconcentration was 23.54 mg/ml (0.25 drug/lipid ratio). The pH of thesolution containing the formulation was 7.31. The empty liposomes had anaverage size of 66.8 nanometers with 92.05 mg/ml of lipid. Theconcentration of the free amikacin solution was 23.54 mg/ml. The pH ofthe solution containing the liposomes was 7.33.

Mice were treated with 40 mg/kg (drug per body-weight), 80 mg/kg, 120mg/kg and 240 mg/kg of liposomal amikacin. Mice were also treated with40 mg/kg, 80 mg/kg, and 120 mg/kg of free amikacin. The drugs wereadministered intravenously in the caudal vein. Two doses were given; oneat four hours after infection and one at twenty-four hours afterinfection. The results are listed in Table 4. The results establish thatit is possible to deliver up to 240 mg/kg of amikacin using a liposomeformulation with all the subjects surviving.

                  TABLE 4                                                         ______________________________________                                        Effects of Liposomal Amikacin on Pseudomonas Infection in Mice                              Mortality                                                                     #died/#tested hours post infection                              Infection                                                                            Treatment    24     30   48   72   %                                   ______________________________________                                        no     none         0/4    0/4  0/4       0                                   yes    none         5/6         6/6       100                                 yes     40 (free amikacin)                                                                        1/4    1/4  1/4  1/4  25                                  yes     80 (free amikacin)                                                                        0/6    0/6  0/6  0/6  0                                   yes    120 (free amikacin)                                                                        0/6    0/6  0/6  0/6  0                                   yes     40 (lipo. amikacin)                                                                       1/4    3/4  4/4  4/4  100                                 yes     80 (lipo. amikacin)                                                                       0/6    0/6  1/6  2/6  33                                  yes    120 (lipo. amikacin)                                                                       0/6    0/6  0/6  0/6  0                                   yes    240 (lipo. amikacin)                                                                       0/6    0/6  0/6  0/6  0                                   ______________________________________                                    

EXAMPLE 5

Amikacin content was measured in mouse (C57B1/6, females 2-3 months old)plasma after injection (i.v.) of free amikacin or liposomal amikacin(100 mg/kg). Liposomal amikacin was prepared as described in Example 2.Blood samples were obtained at various time intervals. The samples werecollected by removing 100 microliters of blood, retroorbitally, fromnonanesthetized mice. The samples were collected in heparinizedcapillary pipets which were plugged with modeling clay. The samples werecentrifuged at 3250 rpm (20 cm rotor) for 10 minutes. The samples wereanalyzed using a radioimmunoassay (Diagnostic Products). The liposomalamikacin formulation contained liposomes with an average size of 43.6 nm(median diameter). The amikacin concentration was 10.09 mg/ml and thetotal lipid concentration was 56.8 mg/ml (drug to lipid ratio 1:5.556).The results are listed in Table 5.

                  TABLE 5                                                         ______________________________________                                        Amikacin Content in Mouse Plasma                                                                          Average  Standard                                                             Concentration                                                                          deviation                                Treatment                                                                             Interval (hours)                                                                         # of mice                                                                              (μg/ml)                                                                             (±μg/ml)                           ______________________________________                                        Amikacin                                                                              0.0833     4        406      155                                              0.25       4        106       12                                               2         4         65       12                                               6         4        none detected                                             14                  not tested                                                24                  not tested                                        Liposomal                                                                             0.0833     4        530      258                                      Amikacin                                                                              0.25       4        344      321                                               2         3        346      199                                               6         4        278      137                                              14         4        272       78                                              24         6        108       14                                      ______________________________________                                    

The results establish that liposomal amikacin is retained in the plasmafor a significantly longer period than free amikacin.

Although this specification has been disclosed and illustrated withreference to particular applications, the principles involved aresusceptible to numerous other applications which will be apparent tothose skilled in the art. The invention is, therefore, to be limitedonly as indicated by the scope of the appended claims.

EXAMPLE 6

Liposome encapsulated amikacin was tested against both drug resistantand drug susceptible strains of M. tuberculosis. Liposomes were preparedas in Example 1.

Two test strains of M. tuberculosis were identified by the Gen-Pro (SanDiego) technique. One strain was a drug resistant strain known asVertulla. The other strain is known as H37RV. Both strains are locatedat the National Jewish Center for Immunology and Respiratory Medicine,Denver, Colo. For each experiment, a subculture in 7H9 broth was madefrom a frozen aliquot. After 1 to 2 weeks of incubation at 37° C. on aconstantly rotating roller drum, the bacterial suspension for eachstrain was forced through a 27-gauge needle and then centrifuged at250×g for 5 minutes to remove large clumps of bacteria. Peripheral bloodfrom healthy purified protein derivative-negative donors was collectedin a 60 ml syringe that had been pretreated with approximately 0.06 mlof a preservative-free solution containing 10,000 IU of heparin per ml(GIBCO Laboratories, Grand Island, N.Y.). This pretreated resulted in 10IU/ml of blood. The 60 ml blood sample was placed in a tube containing aFicoll-Hypaque gradient (Sigma Diagnostics, St. Louis, Mo.) andcentrifuged at 800×g for 15 minutes at room temperature, in accordancewith the manufacturer's instructions. The mononuclear cell band wascollected, transferred to a 50 ml Falcon tube, and diluted with RPMI1640 (GIBCO), containing 10 IU of heparin per ml, to a total volume of40.0 ml. The suspension was centrifuged at 250×g for 10 minutes at roomtemperature. The cells were washed once in 10 ml of RPMI 1640 containingheparin and the pellet was resuspended in RPMI 1640 and adjusted to 10⁷cells per ml. The suspension was placed in 35 mm plastic petri plates(Becton Dickinson Labware, Lincoln Park, N.J.) in two spots, 1 drop(approximately 0.05 ml) for each, resulting in monolayers containingabout 5×10⁵ cells. The plates were incubated at 37° C. for 1 hour toallow the cells to adhere and were then washed twice with RPMI 1640.Then, 1.5 ml of RPMI 1640 containing 3% human autologous nonheated freshserum was added to each plate for a 7 day period of incubation at 37° C.in the presence of 7% CO₂. The pH of the medium was 7.2 to 7.3.

The above-described bacterial suspension was centrifuged at 3,500×g for30 min in a refrigerated centrifuge, and the pellet was resuspended in2.0 ml of RPMI 1640. To estimate the number of acid-fast bacilli permilliliter of this suspension, 0.01 ml samples of the suspension wereplaced on Reich counting slides (Bellco Biotechnology, Vineland, N.J.)with a known number of fields (under ×1,000 magnification) per circle.The slides were fixed and stained. The numbers of acid-fast bacilli permilliliter estimated from the counts on these slides proved to beaccurate, as shown previously. On the basis of these counts, thebacterial suspension in RPMI 1640 was adjusted to contain 10⁶ acid-fastbacilli per ml.

After 7 days of incubation, the monocytes were considered to havematured into a macrophage monolayer. The medium was removed from theplates and replaced with the bacterial suspension at 1.5 ml per plate.After incubation for one hour, the plates were washed three times toremove the extracellular bacteria. The infected macrophages wereincubated in RPMI 1640 supplemented with 1% human autologous serum.Liposomal and free amikacin was added to the cultures at the followingconcentrations (μg/ml of RPMI): 1, 2 and 4 for susceptible strain and 4,8 and 16 for resistant strain. Drug free liposomes and untreatedcultures were used as controls. The experiment was carried out induplicate. The bacterial counts used in the experiment described belowrepresented only intracellular bacteria. At days 0, 4, and 7, the mediumfrom alternate plates was discarded, and the monolayers were lysed byexposure for 10 minutes to a 0.25% solution of sodium dodecyl sulfate at1.0 ml per plate. After the suspension was transferred to a tube, theplate was rinsed with 1.0 ml of 7H9 broth containing 20% bovine albumin,and the rinse was then added to the same tube. Tenfold serial dilutionswere made to inoculate 7H11 agar plates for subsequent colony counts.The results were expressed as the number of cfu per monolayer. Theinitial cfu (at day 0) is shown as measured for the liposomes only(4.3×10³). The results of the experiment are shown in Tables 6 &

                  TABLE 6                                                         ______________________________________                                        Activity of Amikacin (Liposomal and Free) Against Drug Resistant TB           (Vertulla)                                                                             0           4           7                                                              Avg.          Avg.        Avg.                                       CFU      log    CFU    log  CFU    log                               DAYS     (1 × 10.sup.3)                                                                   CFU    (1 × 10.sup.3)                                                                 CFU  (1 × 10.sup.3)                                                                 CFU                               ______________________________________                                        No Treatment             15, 30 4.4  500, 500                                                                             5.7                               Liposomes                                                                              4.6, 4.0 3.6    10, 22 4.2  240, 360                                                                             5.5                               Only                                                                          Liposomal                                                                     Amikacin                                                                      4 μg/ml               1, 1.2 3.0  4, 3.5 3.6                               8 μg/ml               0.2, 0.5                                                                             2.5  0.28, 0.62                                                                           2.7                               16 μg/ml              0.05, 0.03                                                                           1.6  0.08, 0.09                                                                           1.9                               Amikacin                                                                      4 μg/ml               9.3, 8.0                                                                             3.9  210, 100                                                                             5.2                               8 μg/ml               5, 4   3.7  4.5, 6.2                                                                             3.7                               16 μg/ml              5, 5.4 3.7  3.7, 4.1                                                                             3.6                               ______________________________________                                    

The results in Table 6 clearly indicate that liposomal amikacin providesan effective means for treating drug resistant M. tuberculosis. Thelevel of activity against the growth of the drug resistant strain is aleast about 8 times greater than free amikacin and provides activity upto at least 130 times greater than free amikacin at high doses (16μg/ml). Liposomal amikacin at the doses studied obtained killing at eachconcentration whereas the free amikacin obtained, at the most,inhibition.

                  TABLE 7                                                         ______________________________________                                        Activity of Amikacin (Liposomal and Free)                                     Against Drug Susceptible TB                                                          0           4            7                                                             Avg.           Avg.         Avg.                                     CFU      log    CFU     log  CFU     log                               DAYS   (1 × 10.sup.3)                                                                   CFU    (1 × 10.sup.3)                                                                  CFU  (1 × 10.sup.3)                                                                  CFU                               ______________________________________                                        No                     110, 200                                                                              5.2  1700, 1000                                                                            6.1                               Treatment                                                                     Liposomes                                                                            8, 10    4.0    80, 90  4.9  1100, 1300                                                                            6.1                               Only                                                                          Liposomal                                                                     Amikacin                                                                      1 μg/ml             6, 7    3.8  100, 100                                                                              5.0                               2 μg/ml             2, 1    3.2  7.5, 8.6                                                                              3.9                               4 μg/ml             1, 1    3.0  2, 2.5  3.4                               Amikacin                                                                      1 μg/ml             50, 60  4.7  900, 800                                                                              5.9                               2 μg/ml             20, 20  4.3  40, 50  4.7                               4 μg/ml             4,5     3.7  1.5, 3  3.4                               ______________________________________                                         The results in Table 7 show that liposomal amikacin is as effective or        even more effective of inhibiting or killing M. tuberculosis at equivalen     doses.                                                                   

The results in Table 7 show that liposomal amikacin is as effective oreven more effective of inhibiting or killing M. tuberculosis atequivalent doses.

What is claimed:
 1. A composition for treating a bacterial infection ina patient consisting essentially of an aminoglycoside, encapsulated inliposomes, wherein the liposomes are comprised of cholesterol, a neutralamphiphilic lipid and a negatively charged amphiphilic lipid.
 2. Thecomposition of claim 1 wherein the neutral and negatively chargedamphiphilic lipids are saturated.
 3. The composition of claim 2 whereinthe saturated neutral and negatively charged amphiphilic lipids arephospholipids.
 4. The composition of claim 3 wherein the saturatedneutral phospholipid is selected from the group consisting ofhydrogenated egg phosphatidylcholine (HEPC),dimyristoylphosphatidylcholine (DMPC), hydrogenated soyphosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC), anddipalmitoyl phosphatidylcholine (DPPC).
 5. The composition of claim 3wherein the saturated neutral phospholipid is hydrogenated soyphosphatidylcholine (HSPC).
 6. The composition of claim 3 wherein thesaturated negatively charged phospholipid is selected from the groupconsisting of hydrogenated soy phosphatidylglycerol (HSPG), hydrogenatedegg phosphatidylglycerol (HEPG), distearyolphosphatidylglycerol (DSPG),dimyristoyl phosphatidylglycerol (DMPG), anddilaurylphosphatidylglycerol (DLPG).
 7. The composition of claim 3wherein the saturated negatively charged lipid isdistearoylphosphatidylglycerol (DSPG).
 8. The composition of claim 1wherein said aminoglycoside is selected from the group consisting ofstreptomycin, neomycin, kanamycin, gentamicin, tobramycin, sisomicin,amikacin, and netilmicin.
 9. The composition of claim 1 wherein saidaminoglycoside is amikacin.
 10. A composition for inhibiting bacterialgrowth in a patient consisting essentially of an aminoglycosideencapsulated in liposomes, wherein the liposomes are comprised ofcholesterol, a neutral amphiphilic lipid and a negatively chargedamphiphilic lipid, wherein the neutral amphiphilic lipid, cholesteroland negatively charged amphiphilic lipid are in a molar ratio of about2:1:0.1, wherein the aminoglycoside to total lipid molar ratio is from1:9 to 1:3 and wherein said liposomes consist of unilamellar vesicleshaving an average size of less than 100 nm.
 11. A composition fortreating a bacterial infection in a patient consisting essentially ofamikacin encapsulated in liposomes, wherein the liposomes are comprisedof cholesterol, HSPC, and DSPG wherein HSPC:cholesterol:DSPG are in amolar ratio of about 2:1:0.1 wherein the amikacin to total lipid molarratio is from 1:9 to 1:3 and wherein said liposomes consist ofunilamellar vesicles having an average size of less than 100 nm.